The present invention pertains generally to a diagnostic system and/or method, and more particularly to a substantially simultaneous and multiplexed, multi-analyte diagnostic system and/or method for performing assays using a flow analyzer.
Flow cytometry utilizes an optical technique that analyzes particles in a fluid mixture based on the particles"" optical characteristics using a flow cytometer. Background information on flow cytometry is, for example, found in Shapiro, Practical Flow Cytometry, Third Ed. (Alan R. Liss, Inc. 1995), incorporated herein by reference.
Conventional flow cytometers have been commercially available since the early 1970s and presently cost, for example, more than $120,000. They can be behemoths in size, occupying upwards of 13 cubic feet and weighing well over 200 pounds.
In conventional flow cytometers, as shown in FIGS. 1 and 2, sample fluid containing sample cells or microspheres having reactants on their surfaces is introduced from a sample tube into the center of a stream of sheath fluid. The sample fluid stream is injected into, at, or near, the center of the flow cell or cuvette. This process, known as hydrodynamic focusing, allows the cells to be delivered reproducibly to the center of the measuring point. Typically, the cells or microspheres are in suspension in the flow cell.
A continuous wave laser 1900 focuses a laser beam on them as they pass through the laser beam by a flow of a stream of the suspension. Lasers in conventional flow cytometers often require shaping a round beam into an elliptical beam to be focused on the flow cell. As shown in FIG. 2, this elliptical beam is often formed from the round beam using a beam shaping prismatic expander 1960 located between the laser and the flow cell.
When an object of interest 1905 in the flow stream is struck by the laser beam, certain signals are picked up by detectors. These signals include forward light scatter intensity and side light scatter intensity. In the flow cytometers, as shown in FIGS. 1 and 2, light scatter detectors 1930, 1932 are located opposite the laser (relative to the cell) to measure forward light scatter intensity, and to one side of the laser, aligned with the fluid-flow/laser beam intersection to measure side scatter light intensity.
In front of the forward light scatter detector 1930 can be an opaque bar 1920, called a beam stop, that blocks incident light from the laser. Thus, the beam stop ensures that as little of the beam as possible will interfere with the measurement by the forward light scatter detector of the relatively small amount of light which has been scattered, by the flow cell, at small angles to the beam. Forward light scatter intensity provides information concerning the size of individual cells, whereas side light scatter intensity provides information regarding the relative size and refractive property of individual cells.
Known flow cytometers, such as disclosed in U.S. Pat. No. 4,284,412 to HANSEN et al., incorporated herein by reference, have been used, for example, to automatically identify subclasses of blood cells. The identification was based on antigenic determinants on the cell surface which react to antibodies which fluoresce. The sample is illuminated by a focused coherent light and forward light scatter, right angle light scatter, and fluorescence are detected and used to identify the cells.
As described in U.S. Pat. No. 5,747,349 to VAN DEN ENGH et al., incorporated herein by reference, some flow cytometers use fluorescent microspheres, which are beads impregnated with a fluorescent dye. Surfaces of the microspheres are coated with a tag that is attracted to a receptor on a cell, an antigen, an antibody, or the like in the sample fluid. So, the microspheres, having fluorescent dyes, bind specifically to cellular constituents. Often two or more dyes are used simultaneously, each dye being responsible for detecting a specific condition.
Typically, the dye is excited by the laser beam from a continuous wave laser 1900, and then emits light at a longer wavelength. As shown in FIG. 1, dichroic filters 1940 split this emitted light and direct it through optical detectors 1950, 1952, 1954 that can be arranged relative to the laser. The optical detectors 1950, 1952, 1954 measure the intensity of the wavelength passed through, respective filter. The fluorescence intensity is a function of the cells"" absorption of fluorescent dye.
FIG. 2 depicts a prior art flow cytometer which uses beam splitters 1942, 1944, 1946 to direct light from the flow cell 1910 to photo-multiplier and filter sets 1956, 1958, 1959 and to side light scatter detector 1932. This flow cytometer employs a mirror 1970 to reflect forward light scatter to forward light scatter detector 1930.
However, I have determined that the properties of the fluorescent dyes themselves limit this flow cytometric technique to about three different wavelengths. The difference in energy, and hence wavelength, between an excitation photon and emission photon is known as Stokes shift. Generally, the larger the Stokes shift from the excitation wavelength, the broader and weaker the emission spectra
At any given excitation wavelength, I have determined that there are often only a limited number of dyes that emit a spectrum of wavelengths narrow enough and sufficiently separated enough that they are individually measurable simultaneously. Of these, there are fewer dyes still that exhibit good quantum efficiency, for example, between 5and 40%. Other values for quantum efficiency are also acceptable. For example, values of 75 to 80% are acceptable. Consequently, researchers in flow cytometry and other fields have been limited to roughly three fluorescent labels, namely, for green, yellow-orange, and red light.
The limitation on the number of fluorescent labels necessarily crimps the amount of analysis that can be done on any one sample. Therefore, for meaningful analysis, a larger quantity of sample is required and more runs of the sample through the flow cytometer must be performed. This necessarily increases the time needed to analyze the sample. However, time is often not available in an emergency room environment, for example, where a small blood sample, must be screened simultaneously for many diagnostic indicators, including therapeutic and abused drugs, hormones, markers of heart attack and inflammation, and markers of hepatic and renal function. In addition, for efficiency reasons, it is desirable to minimize the testing time to increase the number of tests that can be performed over a predetermined time interval.
One way to overcome the limitation on the number of fluorescent labels, I have determined, is to use two lasers of different frequencies, each focused on a different spot along the flow stream. Such a configuration is called a multi-station flow cytometer. As a particle passes a first laser, up to three fluorescence measurements are taken. Then, as the particle passes the second laser, up to three more measurements are taken using a time-gated amplifier at a predetermined time interval after signals have been detected at the upstream observation point. FIG. 3 illustrates this method.
It should be noted that the upper pair of particles A, B show the lower pair of particles A, B at a later time as the particles progress upward through the flow cell; the particles themselves are the same. In this case, laser #1 strikes particle A. A detector for Laser #2 must wait for a particle to pass through the beam of Laser #2.
Despite this dual laser approach, I have determined that it is often impossible to know for certain whether the measurements are made on the same particle. Because the measurement events at the sets of detectors are separated temporally and spatially, I have discovered that, besides laser emission timing problems, even the slightest flow turbulence can mix particles in suspension, thereby increasing the likelihood that subsequent measurements are not made on the same particle as the previous measurements.
Further, particles in the sample fluid exhibit different velocities as they pass through the flow cell depending on their respective distances from the center of the sample fluid flow stream. Plainly, a an particle closer to the center would travel faster than a particle further away from the center. As such, it is difficult or impossible to be sure exactly when a particle detected by a detector for Laser #1 will pass through a beam of Laser #2.
Referring to FIG. 3, flow turbulence, for example, causes particle B to change places with particle A such that laser #2 strikes particle B, instead of particle A. By extension, this unacceptable problem compounds as lasers and detectors are added to the device.
Despite this flaw, such multiple illumination beam capabilities have been limited to expensive, complex sorters and are not typically found in smaller, less expensive instruments. Besides being large and expensive, such machines are often fully burdened in the clinical setting with CD4-CD8 lymphocyte analysis.
Compounding the above-mentioned shortcomings of existing devices and methods, I have discovered that existing methods of data collection and analysis thereof is tedious, slow, and non-real-time. That is, substantially simultaneous detection of multiple analytes, or of separately identifiable characteristics of one or more analytes, through single-step assay processes is presently not possible, or to the extent possible, has provided limited capability and thus has yielded unsatisfactory results. Reasons for these disappointing results include the following. First, the length of time typically required to enable detection and classification of multiple analytes is unacceptably long. Second, the prior art assays exhibited low analyte sensitivities, which often lead to significant analytical errors and unwieldy collection, classification, and analysis of prior art algorithms relative to large amounts of collected data.
An existing bead set separation method involves the following steps. First, a test tube having sample fluid and sets of reporter beads must be loaded into the flow cytometer and depress the xe2x80x9cAcquirexe2x80x9d button. Second, when the desired number of data events have been collected, the xe2x80x9cStopxe2x80x9d button must be pressed. Third, a file containing the collected data must be saved to a hard drive of a computer. Fourth, a control and analysis software package must be opened. Fifth, the file must be loaded into the control and analysis software package. Sixth, an x-y plot of FL2 v. FL3 must be charted, where FL2 and FL3 are orange and red fluorescence classification parameters for the sets of beads. Seventh, the sets of beads in the plot, represented by clouds of dots, must be visually located and a polygon gate must be drawn around the first set of interest to eliminate stray data points. Eighth, the file must be filtered for events that fall within the polygon gate. Ninth, the statistics must be displayed and the mean value of FL1 must be noted, wherein FL1 is the green fluorescence measurement for the analyte of interest. Tenth, FL1 to FL2 percent spill-over must be calculated and subtracted from the mean value of FL2 to correct the value of FL2. Eleventh, the corrected value of FL2 is used to look up manually which bead set was located in the polygon gate. Twelfth, the FL2 to FL1 percent spill-over is calculated and subtracted from the mean value of FL1. Thirteenth, the assay result is determined from the adjusted value of FL1. The previous thirteen steps are manually repeated for each remaining set of beads.
In addition to the tedium associated with the above-described bead set separation method, I have discovered that the subjectivity associated with estimating the boundaries of the polygon gates is unacceptable. The value of any assay using this method depends largely on the variable judgment of a lab technician. It is often impossible to separate some sets of beads because of overlap of bead regions on the FL2-FL3 plot. Moreover, because of FL1 to FL2 spill-over, the FL2 value of a subset increases sufficiently to overlap with other fluorescence values of other bead sets. Consequently, because of the spill-over, two subsets occupy substantially the same region, making them impossible to distinguish visually there between. The net result of these difficulties is the inability to determine during a sample run, the existence and quantity of an analyte of interest.
In view of the above, I have determined that it would be desirable to have a system and/or method for detecting multiple analytes in a fluid sample by flow cytometric analysis and for analyzing and presenting the data in real-time.
I have determined that it would be desirable to have such a system and/or method, which eliminates the variability of human judgment and subjectivity from the data collection and analysis by performing data collection, bead set classification, and analysis techniques all carried out substantially simultaneously or contemporaneously.
I have also determined that it would be desirable to have such a system and/or method using a flow analyzer that is a fraction of the size, weight, and cost of conventional flow cytometers. That is, I have determined that the current xe2x80x9cmainframe-stylexe2x80x9d flow cytometer must be replaced by a xe2x80x9cdesktop-stylexe2x80x9d personal cytometer.
I have further determined that it would be desirable to have such a system that is many times as fast as conventional flow cytometers and yet requires a fraction of the sample volume demanded by the conventional flow cytometers.
I have also recognized a deficiency in the current approach to signal processing in flow cytometry, which uses peak detectors to measure an event. When a peak is found, the peak detectors are disabled while the peaks are measured and processed. xe2x80x9cDead time,xe2x80x9d the time period during which events can pass through the laser focal point undetected, is highly problematic when the flow cytometer is being used to search for rare events.
Prior art methods, such as U.S. Pat. No. 5,550,058 to Corio et al., incorporated herein by reference, are largely unsuccessful. However, no known prior art method and/or system, including that of Corio et al., has reduced dead time to zero. For example, Corio et al. pre-qualifies an event electronically to reduce the chance that a rare event slips by during dead time. The Corio et al. system sorts particles at a selected yield/purity ratio which ratio can include an intermediate value of the maximum yield and the maximum purity.
Prior art systems and/or methods, which do not use peak detection, use an integrator to measure the area under the pulse. Again, events pass through the laser beam undetected while the measurement is made. Thus, use of an integrator also fails to reduce dead time to zero.
In view of the above-described dead time problem, I have determined that it would be desirable to have a system and/or method for detecting multiple analytes in a fluid sample that reduces dead time in flow analysis to zero.
It is, therefore, a feature and advantage of the instant invention to provide a system and/or method for detecting multiple analytes in a fluid sample by flow cytometric analysis and for analyzing and presenting the data in real-time.
It is also a feature and advantage of the present invention to provide such a system and/or method, which eliminates the variability of human judgment and subjectivity from the data collection and analysis by performing data collection, bead set classification, and analysis techniques, substantially simultaneously or contemporaneously.
It is another feature and advantage of the instant invention to provide such a system and/or method using a flow analyzer that is a fraction of the size, weight, and cost of conventional flow cytometers. That is, I have determined that to deliver the maximum benefit of the instant diagnostic system to the greatest number of users, the current xe2x80x9cmainframe-stylexe2x80x9d flow cytometer must be replaced by a xe2x80x9cdesktop-stylexe2x80x9d personal cytometer.
It is also a feature and advantage of the present invention to provide such a system that is many times as fast as conventional flow cytometers and yet requires a fraction of the sample volume demanded by the conventional flow cytometers.
It is also a feature and advantage of the present invention to provide such a system and/or method that reduces dead time to zero. For example, the system and/or method include constant fixed rate over sampling where signal samples are continuously stored at a predefined interval. By using a second thread to analyze the contents of the circular buffer and process the events, events are never missed, and, hence, there is no dead time.
To this end, it is a feature and advantage of the instant invention to provide a system including a flow analyzer that is approximately one-eighth the size, weight, and cost of most conventional flow cytometers. The system, optionally, is approximately eight times as fast, and, for example, requires one-eighth the sample volume. The system, optionally, is modular to facilitate easy on-site repair and component upgrade. Optionally, it is also controlled by an industry-standard serial or parallel interface, allowing the system to run on a variety of, for example, personal computer environments and to form laptop or desktop factors, under the direction of, for example, a user-friendly graphical user interface.
By achieving the specifications described above, the instant invention provides heretofore uncommon applications for multi-analyte diagnostic systems, ranging from a large clinical laboratory to small point-of-care facility. That is, the speed and technical elegance of the system make it well-suited to, for example, an emergency room environment, where a small blood sample, for example, is screened simultaneously for many diagnostic indicators. Such indicators, for example, include therapeutic and abused drugs, hormones, markers of heart attack and inflammation, and/or those of hepatic and renal function.
The small size, low cost, and quiet operation of the system allows placement thereof in, for example, virtually every blood bank. Donors at such an equipped blood bank can be tested instantly for blood type and transmissible infectious diseases, thereby advantageously avoiding the collection of blood units destined for rejection. Additionally, the small sample volumes processed by the instant system bring the power of multi-analyte testing to, for example, neonatal and pediatric clinics, often advantageously performing complex analyses for less than the cost of a single analyte conventional test.
In an exemplary embodiment, the instant multi-analyte diagnostic system performs real-time bioassays using, for example, multiple classes of microspheres. Each microsphere in a class is coated with a reactant unique to that class. Each class, for example, serves to assay for a respective analyte of interest. Alternatively, more than one class of microspheres, for example, serves to assay for the same analyte of interest. The classes, optionally, are distinguishable by fluorescent labels and/or size so that each class has a respective color and/or size signature. Thus, using the multiple classes of microspheres, multiple analytes, for example, are assayed simultaneously.
The reactants of these assays, for example, are anchored or secured to the surface of the above-mentioned uniquely fluorescent microspheres. Each assay includes at least one microsphere, and preferably up to a thousand or more microspheres. Thus, for example, to conduct one hundred assays, the instant invention includes, for example, one hundred distinguishable classes of microspheres, totaling, for example, 100,000 microspheres. The instant invention, for example, individually analyzes each microsphere in a flow stream at a rate of up to 20,000 or more beads per second, accurately classifying each to its own unique class or subset based on its fluorescent color and/or size signature. Additionally, the instant invention scans each microsphere for the presence of a color, different from those used to provide class signatures, that quantifies the assay occurring at the surface of each microsphere.
By way of illustration, application of the instant invention is, for example, found in an allergist""s office. An allergist, for example, screens a patient for various allergic sensitivities. Current methods require that a patient""s blood sample be sent from the office to a large clinical laboratory, or that a standard xe2x80x9cscratchxe2x80x9d test be performed on a patient""s skin. Plainly, waiting for blood test results from a large clinical laboratory necessarily limits immediate patient care.
Skin testing patients, using the xe2x80x9cscratchxe2x80x9d test, is used for suspected immediate-type hypersensitivity to one or more environmental substances. The test is performed by placing a drop of allergen(s) on the skin and making a needle prick through the drop(s) and into the underlying epidermis. Puncture sites are examined over the next 20 minutes for a wheal and flare skin response which, if present, indicates antibody-mediated (IgE) hypersensitivity to the test allergen. The scratch test is subject to an unacceptable rate of false-positives, false-negatives, and limited sensitivity.
In contrast, the instant invention, optionally, incubates, for example, a single drop, or more than a drop, of patient blood for less than fifteen minutes, between fifteen to thirty minutes, or greater than thirty minutes. Then, running the incubated sample through the instant invention in a matter of seconds, the diagnostic system provides a highly accurate, quantitative analysis, and if desired, a qualitative analysis, of hypersensitivity to, for example, the sixty-four allergens simultaneously or substantially simultaneously. In these assays, the reagent or reactant used is, for example, 0.1% or less than that required for a conventional enzyme linked immunosorbent assay (ELISA) format.
More specifically, the instant invention provides a multi-analyte diagnostic system for use with a computer. The diagnostic system, for example, includes a flow analyzer including, a substantially co-planar optical assembly having at least one light source and at least one optical detector. The flow analyzer is, optionally, communicatable with the computer. The diagnostic system, optionally, also includes a memory medium readable by the computer and storing computer instructions. The instructions, for example, include the following sequential, non-sequential, or independent steps. A biological sample, for example, is run through, or processed using, the flow analyzer. An identity and quantity of one or more analytes of interest in the biological sample, for example, is determined substantially simultaneously to the running or processing step. The one or more light sources optionally include a plurality of light sources and the one or more optical detectors optionally include a plurality of optical detectors. The plurality of light sources includes identical, similar, or overlapping focal regions. The plurality of light sources, for example, includes a plurality of laser diodes emitting continuous wave light. The plurality of laser diodes optionally includes laser diodes emitting a plurality of wavelengths of continuous wave light. Optionally, the laser diodes include one or more diode pumped lasers, such as YAG lasers.
The flow analyzer, optionally, includes a cuvette having a flat air-to-glass interface relative to each light source and relative to each optical detector. The cuvette, optionally, includes a cuvette having a hexagonal cross-section. Optionally, the cuvette includes a substantially flat glass-to-fluid interface. The cuvette optionally includes a neck region having one of an internal rectangular cross-section and an internal square cross-section.
The one or more light sources, optionally, include two light sources. Each light source, optionally, emits respective two distinct wavelengths of light. The one or more optical detectors, optionally, includes four optical detectors.
The flow analyzer, optionally, includes a multi-pass filter or a plurality of bandpass filters optically coupled in parallel to one or more optical detectors via a respective multi-mode cable. The flow analyzer, optionally, includes, for each band-pass filter, a standard amplifying photo-detector and a standard analog-to-digital converter connected in series thereto. The amplifying photo-detector includes a standard photomultiplier tube, a standard avalanche photo-diode, or a standard p-i-n photo-diode. The flow analyzer, for example, includes for each band-pass filter, an optional standard inverting amplifier in series with a standard low pass Nyquist filter, connected between the amplifying photo-detector and the analog-to-digital converter.
The flow analyzer, optionally, includes one or more magnification lens for magnifying light emission or reflection from the cuvette. For example, the magnification lens may include a lens having a magnification of up to or more than 15xc3x97. Advantageously, the magnification lens obviates use of the above-mentioned multi-mode cable or optical fiber. For example, the flow analyzer optionally-includes a mirror reflecting the light from the cuvette to appropriate detectors.
The diagnostic system optionally includes a digital interface board in the flow analyzer and connectable to the computer via a serial or parallel interface. The digital interface board, optionally, includes a standard microcontroller in communication with the flow analyzer, and a standard digital signal processor in communication with the microcontroller and each analog-to-digital converter. The digital signal processor, optionally, includes a standard circular memory buffer having a first movable pointer, a second movable pointer, and a plurality of storage positions. The first pointer, optionally, points to an oldest storage position into which new sample data can be stored. The second pointer, optionally, points to a storage position from which the digital signal processor is to read the next sample data to be analyzed. The flow analyzer includes a cuvette, a sample pump communicating with the microcontroller and connected to the cuvette, and a sheath fluid reservoir communicating with the microcontroller and connected to the cuvette. The flow analyzer, optionally, includes a waste receptacle. The microcontroller, upon assay completion, optionally, communicates with the sample pump to halt sample fluid flow or divert any remaining sample to the waste receptacle, and, optionally, communicates with the sheath fluid reservoir to halt sheath fluid flow or divert any remaining sheath fluid to the waste receptacle. Optionally, the flow analyzer includes a single-filter light path from each optical detector to each amplifying photo-detector.
The diagnostic system, optionally, further includes a vertically and/or horizontally moveable platform. The flow analyzer, optionally, includes a vertically moveable aspirator. The platform, optionally, cooperates with the aspirator. The platform, optionally, supports a microtiter plate for the flow analyzer.
The instruction for running the instant flow analyzer, optionally, includes exposing a pooled population of subsets of particles to the biological sample, the particles in each subset having (i) one or more classification parameters that distinguish the particles of one subset from those of another subset, and (ii) a reactant specific for each analyte of interest. The running instruction, optionally, further includes passing the exposed pooled population of subsets of particles through an examination zone.
The instruction for determining the identity and quantity of one or more analytes of interest in a biological sample, optionally, includes assessing the identity and quantity of each analyte of interest, if present, in the sample by substantially contemporaneously performing the following steps. Data is, optionally, collected relating to at least one characteristic classification parameter, including data on fluorescence emission intensities. Data is, optionally, collected relating to a presence or absence of a complex formed between the reactant and an analyte of interest specific to the reactant. Without relying exclusively, if at all, on difference""s in particle size, each particle is classified according to its subset. An amount of complex associated with each subset is quantified. The step of collecting data relating to a presence or absence of a complex includes collecting analyte data on fluorescence emission intensities. The bead subset data and the analyte data optionally exhibit spectral overlap. The classifying step, optionally, includes reducing the spectral overlap sufficiently to identify each bead according to its subset.
The diagnostic system, optionally, further includes a circular memory buffer communicatable with the flow analyzer. The circular memory buffer optionally includes first movable pointer in operation, pointing to a storage position available for storing new data, and a second movable pointer, in operation, pointing to a storage position having unanalyzed data.
It is also a feature and advantage of the instant invention to provide a cuvette holder. The cuvette holder, optionally, includes a cuvette holder top including one or more optional viewing grooves along one of a diameter and a width of the top. The cuvette holder, optionally, further includes a cuvette holder base for cooperating with the top to hold a cuvette. Optionally, the cuvette holder further includes a base frame. The cuvette holder top is secured to the base frame. The cuvette holder also optionally includes a stability bracket secured to the base frame and securing a top of the cuvette.
It is another feature and advantage of the instant invention to provide a computer program product. The computer program product, for example, includes a memory medium. The computer program product, for example, also includes a computer program stored on the memory medium. The computer program, for example, contains sequential, non-sequential, or independent instructions as follows. A biological sample is run through a flow analyzer. The biological sample includes a pooled population of bead subsets. Each bead subset has one or more characteristic classification parameters. The characteristic classification parameters, for example, includes one or more characteristic fluorescence emission intensities. Substantially contemporaneously to the running step, data related to the at least one characteristic classification parameter, for example, including bead subset data on fluorescence emission intensities is collected. Substantially contemporaneously to the running step, data related to the presence or absence of an analyte of interest, including, for example, analyte data on fluorescence emission intensities is collected. The bead subset data and the analyte data optionally exhibit spectral overlap. Substantially contemporaneously to the running step, the spectral overlap is optionally reduced sufficiently to identify each bead according to its subset.
The computer program optionally includes instructions for determining, substantially contemporaneously to the running step, a presence and quantity of one or more analytes of interest in the biological sample.
The computer program, optionally, further includes instructions for providing a simplex analysis application module and/or a multiplexed analysis application module. The computer program, optionally, further comprises or stores instructions for providing a main menu, a results table, a system monitor, a dot plot display including a density dot plot and/or a decaying dot ploy, a histogram tab, an optical amplifier control tab, a color compensation control tab, and/or a doublet discriminator control tab.
It is also a feature and advantage of the instant invention to provide a computer program product for use with a flow analyzer and a computer. The computer program product includes a memory medium and a computer program stored on the memory medium. The computer program contains the following sequential, non-sequential, or independent instructions. A biological sample is processed using, or run through, a flow analyzer. The biological sample includes a pooled population of bead subsets. Each bead subset has one or more characteristic classification parameters. The one or more characteristic classification parameters includes one or more characteristic fluorescence emission intensities. Substantially contemporaneously to the processing step, data, related to the at least one characteristic classification parameter including bead subset, data on fluorescence emission intensities, is collected. Substantially contemporaneously to said processing step, data, related to a presence or absence of an analyte of interest, including analyte data on fluorescence emission intensities, is collected. Substantially contemporaneously to the processing step, an identify and quantity of at least one analyte of interest in the biological sample is determined. The computer program product also includes an application programming interface library interfacing with the flow analyzer and the computer program, in operation. The computer program further includes a mathematics library communicating with the computer program, in operation.
The application programming interface library optionally includes one or more of the following functions: a function for initializing a device interface for the flow analyzer; a function for closing a device session with a flow analyzer; a function for loading a map file for distinguishing between the bead subsets; a function for defining bead subsets to be associated with an assay; a function for acquiring bead statistics of a selected bead subset; a function for copying flow analyzer settings into a user-supplied buffer; and a function for changing the flow analyzer settings.
The computer program product optionally includes one or more of the following functions: a function for initiating acquisition of bead statistics for a current sample loaded on the flow analyzer; a function for ending the acquisition of bead statistics; a function for copying most current bead statistics into a user-supplied buffer; and a function for one of returning and displaying data acquisition statistics.
It is another feature and advantage of the instant invention to provide a multi-analyte diagnostic method having the following sequential, non-sequential, or independent steps. A biological sample is processed using, or run-through, a flow analyzer. The biological sample includes a pooled population of bead subsets. Each bead subset has one or more characteristic classification parameters. The one or more characteristic classification parameters includes one or more characteristic fluorescence emission intensities. Substantially contemporaneously to the processing step, data, related to the at least one characteristic classification parameter including bead subset data on fluorescence emission intensities, is collected. Substantially contemporaneously to the processing step, data related to a presence or absence of an analyte of interest, including analyte data on fluorescence emission intensities, is collected. The bead subset data and the analyte data exhibit spectral overlap. Substantially contemporaneously to the processing step, the spectral overlap is reduced sufficiently to identify each bead according to its subset. Substantially contemporaneously to the processing step, an identify and quantity of at least one analyte of interest in the biological sample is determined.
It is another feature and advantage of the instant invention to provide a management system. The management system includes a file system storing static portions of substantially all data pages in a data site. The system also includes a server communicatably connected to the file system. The server retrieves the static portions of one or more data pages stored by the file system and transmits to a site user the static portions of one or more data pages.
Optionally, the management system further includes a data page generator generating the static portions of substantially all data pages based on the data site for storage in the file system. The management system further includes a dynamic data transmit device to transmit dynamic data to be cooperatively presented with the static portions as the at least one data page to the site user. Optionally, the data page generator generates the static portions of the substantially all data pages and provides corresponding indexes therewith. Optionally, the server transmits the static portions to the site user responsive to the corresponding index associated with the at least one data page.
It is another feature and advantage of the instant invention to provide a method of managing a data site having the following sequential, non-sequential, or independent steps. Static portions of substantially all data pages in a data site, are stored using a file system. The static portions of at least one data page stored by the file system, are retrieved using a server communicatably connected to the file system. The static portions of the at least one data page, are transmitted to a site user using the server.
Optionally, the static portions of the substantially all data pages based on the data site for storage in the file system, are generated using a data page generator. Optionally, dynamic data to be cooperatively presented with the static portions as the at least one data page to the site user, are transmitted using a dynamic data transmit device. Optionally, the data page generator generates the static portions of the substantially all data pages and provides corresponding indexes therewith. Optionally, the server transmits the static portions to the site user responsive to the corresponding index associated with the at least one data page.
It is another feature and advantage of the instant invention to provide an analysis or diagnostic method, having the following sequential, sequence independent, or non-sequential, steps. A plurality of pooled subsets are processed through an inspection area, each of the plurality of pooled subsets including one or, more indication parameters. Each of the plurality of samples are illuminated, substantially simultaneously and not sequentially, with two or more light beams from one or more sources, at substantially the same time. One or more indication parameters are determined responsive to the illuminating step. Optionally, each of the light beams according to this method includes continuous wave light.
It is another feature and advantage of the instant invention to include a novel flow cytometer including a base section. A plurality of light sources is mounted to the base section. A plurality of selectors is mounted to the base section. A sample viewing chamber is mounted to the base section and in optical relationship with the plurality of light sources and the plurality of detectors.
It is another feature and advantage of the instant invention to include an analysis or diagnostic system. The instant diagnostic system according to this embodiment includes an initialization system initializing a device interface for a flow cytometer, including a termination system terminating a device session for the flow cytometer; a bead map file system loading a file defining a bead map indicative of an associated bead type; a reset system resetting beads to be used in the analysis or diagnostic system; and a user bead component system used to acquire bead statistics for the beads and the associated bead type.
The instant diagnostic system according to this embodiment further includes a machine control and monitoring system, responsively coupled to the initialization system, and monitoring and controlling the analysis or diagnostic system, including: a panel setting system maintaining current flow cytometer settings in a buffer or storage area; and a change panel setting system changing at least one of the current flow cytometer settings responsive to a command.
The instant diagnostic system according to this embodiment also includes a sample acquisition and reporting system, responsively coupled to the machine control and monitoring system, collecting data for analysis or diagnosis, including: a test start system indicating when to begin collecting the data from the machine control and monitoring system; a test stop system indicating when to stop collecting the data from said machine control and monitoring system; a test stop system indicating when to stop collecting the data from said machine control and monitoring system; a test storage system storing the data in another buffer or storage area; and a test query system performing the analysis or diagnosis on the data responsive to a predetermined program or user query.
It is another feature and advantage of the instant invention to include an analysis or diagnostic method having the following independent, sequential, or non-sequential steps. A device interface for a flow cytometer is initialized. The initializing step includes terminating a device session for the flow cytometer; loading a bead map file defining a bead map indicative of an associated bead type; resetting beads to be used in the analysis or diagnostic system; and acquiring bead statistics for the beads and the associated bead type.
An analysis or diagnosis is controlled and monitored using a machine control and monitoring system. The controlling and monitoring step includes: maintaining current flow cytometer settings in a buffer or storage area; and changing at least one of the current flow cytometer settings responsive to a command.
Data is collected for analysis or diagnosis. The data collection step includes: indicating when to begin collecting the data from the machine control and monitoring system; indicating when to stop collecting the data from the machine control and monitoring system; storing the data in another buffer or storage area; and performing the analysis or diagnosis on the data responsive to a predetermined program or user query.
It is another feature and advantage of the instant invention to provide a detector apparatus including a U-block assembly. The detector apparatus includes one or more optical beam splitters and one or more optical detectors secured to the U-block assembly. The detector apparatus further includes one or more push-pull assemblies adjustably securing the one or more optical beam splitters to the U-block assembly, and directing the one or more optical beam splitters to be sufficiently optically couplable with the one or more optical detectors.
Optionally, the U-block assembly includes a unitary or integrated body having an inner portion. The one or more optical beam splitter bordered, in part, by the inner portion of the U-block assembly. The body of the U-block assembly includes first and second legs. The one or more optical detectors are secured within the first leg, and the one or more push-pull assemblies are secured within the second leg. Optionally, the detector apparatus further includes an optical assembly base frame, wherein the U-block assembly is secured thereto. The one or more push-pull assemblies includes a screw tap and spring assembly for pushing a side of the one or more beam splitters toward the one or more optical detectors, and/or pulling another side of the one or more beam splitters away from the one or more optical detectors.
Optionally, the one or more optical beam splitters includes one or more dichroic mirrors. The one or more dichroic mirrors includes a plurality of dichroic mirrors. Each dichroic mirror directs a respective band of wavelengths of light to a respective optical detector and transmitting a remainder of wavelengths of light therethrough. Optionally, the detector apparatus further comprises one or more filters, interposed between the one or more optical beam splitters and the one or more optical detectors.
It is also a feature and advantage of the instant invention to provide, in a flow analyzer including a pressure sensor and a sheath fluid reservoir, a de-bubbler. The novel de-bubbler includes a bottle including an upper portion and a lower portion. The upper portion of the bottle includes an inlet operatively connected to the sheath fluid reservoir to receive sheath fluid therefrom. The lower portion of the bottle includes an outlet operatively connected to the pressure sensor. The bottle further includes a substantially waterproof vent sealing a top of the bottle and exposing an interior of the bottle to an atmosphere external to the bottle so that, in operation, sheath fluid is output via the outlet substantially free of a gas bubble.
Optionally, the flow analyzer further includes a sample pump. The pressure sensor optionally transmits a command to deactivate the sample pump upon sensing a decreased fluid pressure in the de-bubbler.
It is another feature and advantage of the instant invention to provide a multi-analyte diagnostic system for analyzing a sample fluid for one or more analytes of interest. The multi-analyte diagnostic-system includes a flow analyzer, which includes a cuvette including, in operation, a fluid core, and including a neck region having a substantially flat glass-to-fluid interface and a substantially flat air-to-glass interface. The flow analyzer also includes a first magnification lens optically cooperative with the cuvette and having a magnification power. The flow analyzer further includes a filter and optical amplifier assembly including an entrance aperture. The entrance aperture is dimensioned to cooperate with the magnification power to transmit light from the fluid core in the cuvette with substantially no light distortion from the glass-to-fluid interface and/or the air-to-glass interface.
Optionally, the flow analyzer is communicatable with a computer. The multi-analyte diagnostic system optionally includes a memory medium readable by the computer and storing computer instructions executed by the computer. The computer instructions include processing the sample fluid using said flow analyzer, and analyzing the sample fluid and determining a presence and quantity of at least one analyte of interest in the sample fluid substantially simultaneously to the processing step.
Optionally the multi-analyte diagnostic system further includes a first mirror optically coupled to the first magnification lens and reflecting a first plurality of wavelengths of the light to the entrance aperture, at least one of the first plurality of wavelengths indicative of a presence of one or more analytes of interest in the sample fluid.
Optionally, the flow analyzer further includes one or more light sources to radiate the cuvette. The one or more light sources include a laser diode and/or a diode pumped laser.
Optionally, the cuvette includes upper and lower portions. Optionally, the multi-analyte diagnostic system further includes an optical assembly base frame. The first magnification lens, the filter and optical amplifier assembly, and the one or more light sources are secured to the optical assembly base frame. The multi-analyte diagnostic system further includes a cuvette holder secured to the optical assembly base frame and securing a bottom of the cuvette. The diagnostic system also includes an optional stability bracket secured to the optical assembly base frame and securing a top of the cuvette.
Optionally, the multi-analyte diagnostic system further includes a second magnification lens optically cooperative with the cuvette. The diagnostic system optionally includes one or more optical beam splitters optically cooperative with the second magnification lens. The diagnostic system optionally includes one or more optical detectors identifying one or more particles as belonging to a respective particle subset, and optically cooperative with the one or more optical beam splitters.
Optionally, the multi-analyte diagnostic system further includes a second mirror optically coupled to the second magnification lens and the one or more beam splitters. The second mirror reflects a second plurality of wavelengths of light to the one or more optical beam splitters. One or more of the second plurality of wavelengths are indicative of the identity of the one or more particles.
Optionally, the multi-analyte diagnostic system further includes a side scatter optically cooperating with the one or more beam splitters and identifying a doublet.
There has thus been outlined, rather broadly, the more important features of the invention in order that the detailed description thereof that follows may be better understood, and in order that the present contribution to the art may be better appreciated. There are, of course, additional features of the invention that will be described hereinafter and which will form the subject matter of the claims appended hereto.
In this respect, before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and to the arrangements of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting.
As such, those skilled in the art will appreciate that the conception, upon which this disclosure is based, may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.
Further, the purpose of the foregoing abstract is to enable the. U.S. Patent and Trademark Office and the public generally, and especially the scientists, engineers and practitioners in the art who are not familiar with patent or legal terms or phraseology, to determine quickly from a cursory inspection the nature and essence of the technical disclosure of the application. The abstract is neither intended to define the invention of the application, which is measured by the claims, nor is it intended to be limiting as to the scope of the invention in any way.
These together with other objects of the invention, along with the various features of novelty which characterize the invention, are pointed out with particularity in the claims annexed to and forming a part of this disclosure. For a better understanding of the invention, its operating advantages and the specific objects attained by its uses, reference should be had to the accompanying drawings and descriptive matter in which there is illustrated preferred embodiments of the invention.
The detailed descriptions which follow may be presented in terms of program procedures executed on a computer or network of computers. These procedural descriptions and representations are the means used by those skilled in the art to most effectively convey the substance of their work to others skilled in the art.
A procedure is here, and generally, conceived to be a self-consistent sequence of steps leading to a desired result. These steps are those requiring physical manipulations of physical quantities. Usually, though not necessarily, these quantities take the form of electrical or magnetic signals capable of being stored, transferred, combined, compared and otherwise manipulated. It proves convenient at times, principally for reasons of common usage, to refer to these signals as bits, values, elements, symbols, characters, terms, numbers, or the like. It should be noted, however, that all of these and similar terms are to be associated with the appropriate physical quantities and are merely convenient labels applied to these quantities.
Further, the manipulations performed are often referred to in terms, such as adding or comparing, which are commonly associated with mental operations performed by a human operator. No such capability of a human operator is necessary, or desirable in most cases, in any of the operations described herein which form part of the present invention; the operations are machine operations. Useful machines for performing the operation of the present invention include general purpose digital computers or similar devices.